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C282Y Homozygosity Increases Erythrocyte Turnover and Decreases HbA1c—A Population-Based Study

Int. J. Mol. Sci. 2026, 27(5), 2410; https://doi.org/10.3390/ijms27052410

by Rebekka Hillingsø^1,2, Alisa Devedzic Kjaergaard³, Morten Kranker Larsen⁴, Thomas Mandrup-Poulsen⁵

, Henrik Enghusen Poulsen^6,7

, Mathis Mottelson⁴

, Jesper Brix Petersen⁸, Børge Grønne Nordestgaard^6,9,10,11, Hans Carl Hasselbalch^4,6, Stig Egil Bojesen^6,9,10,11, Jens Helby^6,8,10

, Andreas Glenthøj^6,8,†

and Christina Ellervik^{2,6,12,13,*,†}

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Int. J. Mol. Sci. 2026, 27(5), 2410; https://doi.org/10.3390/ijms27052410

Submission received: 13 January 2026 / Revised: 12 February 2026 / Accepted: 26 February 2026 / Published: 5 March 2026

(This article belongs to the Section Molecular Biology)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "C282Y homozygosity increases erythrocyte turnover and decreases HbA1c - A population-based study" aimed to investigate the association between HFE genotypes, erythrocyte turnover, and HbA1c, and if the association with HbA1c was mediated by transferrin saturation, MCHC, MCV, oxidative stress, hsCRP, erythrocyte count, reticulocyte count, or blood donation. After careful reading, I can make the following recommendations:

1) In Abstract:

a- Add the connection between iron overload and oxidative stress to the first sentence for better context.

b- Add a brief explanation in parentheses or simplify to "lifespan"

c- Highlight that erythrocyte turnover per se was not the primary mediator, which is an important and counterintuitive finding.

2) In Introduction:

a- Add a paragraph on diagnostic and therapeutic implications. "Understanding the mechanisms by which HFE affects HbA1c is clinically relevant because HbA1c is widely used for the diagnosis and monitoring of diabetes, and its misinterpretation in C282Y/C282Y carriers could lead to misdiagnosis."

b- Expand the discussion on eryptosis and phosphatidylserine expression, which are mentioned but not sufficiently explained.

c- It should be clearly added that HbA1c reflects the proportion of glycosylated hemoglobin during the average lifespan of the erythrocyte (approximately 120 days). Therefore, if this lifespan decreases, the measurement is skewed downwards.

d- To specify clearly: "We hypothesize that C282Y/C282Y and, to a lesser extent, other HFE genotypes are associated with increased erythrocyte turnover, which reduces HbA1c by shortening the lifespan of erythrocytes available for hemoglobin glycosylation."

3) In Results:

a- Add an explanatory sentence: "The discordant association between reduced HbA1c and elevated plasma glucose in individuals with C282Y/C282Y is particularly noteworthy and suggests that HbA1c may not be a reliable biomarker in this population."

b- Anticipating this explanation in the Results: "Although we expected that a higher reticulocyte count (young erythrocytes with lower hemoglobin glycosylation) would mediate the association, mediation analyses did not confirm this hypothesis."

c- Figure 3 shows elevations in bilirubin and LDH, but limited interpretation is offered in the Results. Please add: "The elevation in bilirubin and LDH in individuals with C282Y/C282Y is consistent with increased erythrocyte destruction, reflecting greater hemolysis or liver damage (also evidenced by elevated ALT), or both."

d- The text mentions "There was no interaction between HFE genotype and sex," but this is a brief mention.

4) In Discussion:

a- Expanding on the Blood Glucose-HbA1c Paradox: While well-recognized, the discussion could elaborate further on its diagnostic implications.

b- The explanation of the "glucose-to-hemoglobin ratio" is interesting but could be clearer.

c- The discussion does not explicitly mention that this is a cross-sectional/observational study. I recommend adding to the text: "It is important to note that this is an observational study in a population-based study. Although our mediation analyses suggest mechanisms, causality cannot be inferred. Prospective or interventional studies (e.g., phlebotomy with serial HbA1c follow-up) would be needed to confirm causality."

d- Further Explanation of H63D Variants: While C282Y/C282Y receives primary focus, the attenuated findings in other H63D variants could be discussed in more detail.

e- Implications for Glycemic Biomarkers: The recommendation regarding OGTT, fructosamine, and glycated albumin is briefly presented at the end. I recommend expanding on this in a separate paragraph with justification.

5) In Materials and Methods:

a- Pregnancy is an exclusion criterion mentioned briefly. Please improve.

b- Unmeasured Covariates: There is no discussion of potential unmeasured confounders (e.g., alcohol consumption, infection status). Please consider adding them in the Limitations section.

c- Erythrocyte Turnover Calculation: Although described in 4.4, the calculation would benefit from discussion of the assumptions.

d- In the tables, Confidence Interval Inclusion: Tables show the mean (SD) but not the 95% CI.

The manuscript deserves publication after incorporating the above recommendations, particularly:
1. Clarification of the proposed mechanisms
2. Expanded discussion of the glucose-HbA1c paradox
3. Practical recommendations for diagnosis in C282Y/C282Y

Author Response

Reviewer-1

Comments and Suggestions for Authors

Answer: Thank you very much for carefully reading our manuscript and providing valuable feedback. We have answered all your questions, and made changes to the manuscript accordingly

1) In Abstract:

a- Add the connection between iron overload and oxidative stress to the first sentence for better context.

Answer 1a. Thank you for the comment. Due to the strict word limit of 200 words, we have inserted a brief connection in the abstract: “Individuals with C282Y/C282Y in the hemochromatosis HFE gene have increased iron levels, which catalyse the formation of reactive oxygen species…”

b- Add a brief explanation in parentheses or simplify to "lifespan"

Answer 2b: We are uncertain to which particular sentence you are pointing. We changed the second sentence of the abstract to: “These individuals may have a disproportionately lower HbA1c due to increased erythrocyte turnover, decreased erythrocyte counts, and/or increased mean corpuscular hemoglobin concentration (MCHC).”

c- Highlight that erythrocyte turnover per se was not the primary mediator, which is an important and counterintuitive finding.

Answer 1c. We added to the abstract “In conclusion, while C282Y/C282Y and other HFE genotypes increased erythrocyte turnover, the disproportionately decreased HbA1c was explained by larger erythrocytes filled with more hemoglobin and removed earlier from the circulation, thus diluting the relative concentration of intracellular glucose per hemoglobin molecule.”

2) In Introduction:

Answer 2a. Thank you for the suggestion which we have inserted in the introduction.

b- Expand the discussion on eryptosis and phosphatidylserine expression, which are mentioned but not sufficiently explained.

Answer 2b: Thank you for the suggestion. We find that the discussion section is better suited to expand on this. We therefore rephrased the paragraph in the discussion to the following:

“Two key features of programmed erythrocyte removal (eryptosis) are erythrocyte shrinkage and membrane phospholipid scrambling driven by Ca²⁺ influx, which causes phosphatidylserine (PS) to move from the inside to the surface of the erythrocytes (ref). This process can be triggered by oxidative stress(ref) and could be a likely mechanism for the decreased erythrocyte count. Thus, unliganded iron in individuals with C282Y/C282Y, and to a lesser extent, other HFE genotypes, participate in the Fenton reaction, creating reactive oxygen species, which we have previously shown (refs). Increased oxidative stress is also associated with a rigid erythrocytic membrane (refs). And finally, phosphatidylserine expression on the surface of erythrocytes has been observed across all HFE mutations (ref)”

Answer 2c. Thank you for the suggestion. We agree that more emphasis on the proposed mechanism in the introduction is needed. Please see our response to your next question.

d- To specify clearly: "We hypothesize that C282Y/C282Y and, to a lesser extent, other HFE genotypes are associated with increased erythrocyte turnover, which reduces HbA1c by shortening the lifespan of erythrocytes available for hemoglobin glycosylation."

Answer 2d: Thank you for the suggestion. We have expanded the proposed mechanism in the hypothesis, which now reads:

“We therefore hypothesized that C282Y/C282Y, and, to a lesser extent, other HFE genotypes are associated with reduced HbA1c due to increased erythrocyte turnover with proportionately more reticulocytes with less hemoglobin available for glycosylation and/or by increased hemoglobin concentration in larger erythrocytes (MCHC and MCV) ) causing diluting of the relative concentration of intracellular glucose per hemoglobin molecule”

3) In Results:

Answer 3a. Thank you for the suggestion which we have inserted in the results.

Answer 3b. Thank you for the suggestion which we have inserted in the results.

Answer 3c. Thank you for the suggestion which we have inserted in the results slightly modified, as we cannot distinguish hemolysis and eryptosis in this study: “The elevation in bilirubin and LDH in individuals with C282Y/C282Y is consistent with increased erythrocyte destruction, liver damage (also evidenced by elevated ALT), or both.”

d- The text mentions "There was no interaction between HFE genotype and sex," but this is a brief mention.

Answer 3d. We have expanded the text which now reads “Across all regression models, there was no interaction (p>0.05) between HFE genotypes and sex, indicating that the effect of HFE genotypes on the biomarkers did not differ between women and men.” We moved this sentence to a new section “sensitivity analyses”.

4) In Discussion:

a- Expanding on the Blood Glucose-HbA1c Paradox: While well-recognized, the discussion could elaborate further on its diagnostic implications.

Answer 4a. We agree. We are preparing a follow-up to this paper, investigating the diagnostic implications. We added:

“Furthermore, it should be investigated if the decreased HbA1c causes a diagnostic delay of diabetes in individuals with C282Y/C282Y.”

b- The explanation of the "glucose-to-hemoglobin ratio" is interesting but could be clearer.

Answer 4b. We added a supporting statement (underlined) to the last part of the sentence so it now reads: “This alteration in the intracellular glucose-to-hemoglobin ratio could reduce the extent of hemoglobin glycation with C282Y/C282Y and other HFE genotypes, contributing to the observed decrease in HbA1c level, as this reflects the proportion of glycosylated hemoglobin over the erythrocyte lifespan of 120 days.”

Answer 4c. Thank you for the suggestion which we have inserted slightly revised in the Discussion:

“This study was an observational cross-sectional population-based study, and while our mediation analyses suggested mechanisms, causality cannot be inferred.”

And

“Due to the glucose-HbA1c paradox, future studies should evaluate phlebotomy with serial HbA1c follow-up in prospective or interventional studies and alternative erythrocyte-independent glycemic biomarkers to HbA1c in individuals with C282Y/C282Y. Examples include the traditional and clinically approved 2-hour oral glucose tolerance testing (OGTT) and fasting glucose which detect impaired glucose tolerance and diabetes(ref), or the newer but not standardized biomarkers fructosamine (glycated serum proteins) (ref) or glycated albumin(ref), which reflect average glucose over the last 2-3 weeks.””

d- Further Explanation of H63D Variants: While C282Y/C282Y receives primary focus, the attenuated findings in other H63D variants could be discussed in more detail.

Answer 4d. We added to the results in to a new section “Other HFE genotypes: “Other HFE genotypes showed a similar but attenuated pattern, such that the differences for C282Y/H63D and H63D/H63D were approximately 30-50% of that for C282Y/C282Y, and the differences for C282Y/NC and H63D/NC were approximately 10-25% of that for C282/C282Y, although 95% CIs were overlapping in some analyses, indicating less precision. Furthermore, only C282Y/C282Y, not other HFE genotypes, were associated with increased p-glucose.”

Answer 4e. We have moved the paragraph to the one before the conclusions in the discussion. We expanded the section, so it now reads:

“Due to the glucose-HbA1c paradox, future studies should evaluate phlebotomy with serial HbA1c follow-up in prospective or interventional studies and alternative erythrocyte-independent glycemic biomarkers to HbA1c in individuals with C282Y/C282Y. Examples include the traditional and clinically approved 2-hour oral glucose tolerance testing (OGTT) and fasting glucose which detect impaired glucose tolerance and diabetes(ref), or the newer but not standardized biomarkers fructosamine (glycated serum proteins)(ref) or glycated albumin(ref), which reflect average glucose over the last 2-3 weeks. Furthermore, it should be investigated if the decreased HbA1c causes a diagnostic delay of diabetes in individuals with C282Y/C282Y.”

5) In Materials and Methods:

a- Pregnancy is an exclusion criterion mentioned briefly. Please improve.

Answer 5a. We added a statement to justify the exclusion of pregnant women: “…due to pregnancy as this cause physiological changes in iron and glucose metabolism that may confound or distort the association between HFE variants, iron metabolism, and HbA1c..”

b- Unmeasured Covariates: There is no discussion of potential unmeasured confounders (e.g., alcohol consumption, infection status). Please consider adding them in the Limitations section.

Answer 5b. Reviewer-2 also made a similar comment. We have now presented a supplementary table with results from a sensitivity analysis including confounders that are not part of the pathway from genotype->iron->biomarker. In a stepwise regression, the model includes age, sex, genotype, cohort, blood donor status, BMI, alcohol consumption, smoking, physical activity, and infection. Results were similar with or without these covariates in the model. We have also added a paragraph to the statistical section explaining this. And we have commented on this in the results section “sensitivity analyses”. Lastly, we have included information on alcohol, physical activity, and infection in table 1 and in the supplementary tables of the cohorts.

c- Erythrocyte Turnover Calculation: Although described in 4.4, the calculation would benefit from discussion of the assumptions.

Answer 5c. We inserted the following in the discussion: “We used the reticulocyte percentage as a proxy for the erythrocyte turnover, but this was not measured with biotin-labeling which is a more accurate measure. The rate of production of erythrocytes with HFE genotypes is unknown but the reticulocyte hemoglobin was higher with C282Y/C282Y and other HFE genotypes in our study, indicating a later release of more mature reticulocytes from the bone marrow.”

d- In the tables, Confidence Interval Inclusion: Tables show the mean (SD) but not the 95% CI.

Answer 5d. As the tables are quite big and displayed in landscape layout and range from the margins of the document, there is not enough space to provide the 95%CI. Furthermore, the 95%CI is based on the standard error and not the standard deviation, and SD provides information on the spread of the distribution of the data. The 95%CI is more appropriate for statistical analyses.

The manuscript deserves publication after incorporating the above recommendations, particularly:
1. Clarification of the proposed mechanisms
Answer 1. We hope the above answers clarify this.

Expanded discussion of the glucose-HbA1c paradox

Answer 2. We added the following to the discussion, “In contrast, p-glucose was increased in individuals with C282Y/C282Y” (1^st paragraph) and “Due to the glucose-HbA1c paradox, future studies should also evaluate alternative erythrocyte-independent glycemic biomarkers to HbA1c in individuals with C282Y/C282Y” (second to last paragraph in the discussion).

Practical recommendations for diagnosis in C282Y/C282Y

Answer 3. We expanded the following to the second to last paragraph: “Due to the glucose-HbA1c paradox, future studies should evaluate phlebotomy with serial HbA1c follow-up in prospective or interventional studies and alternative erythrocyte-independent glycemic biomarkers to HbA1c in individuals with C282Y/C282Y. Examples include the traditional and clinically approved 2-hour oral glucose tolerance testing (OGTT) and fasting glucose which detect impaired glucose tolerance and diabetes(ref), or the newer but not standardized biomarkers fructosamine (glycated serum proteins) (ref) or glycated albumin(ref), which reflect average glucose over the last 2-3 weeks.”

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript “C282Y Homozygosity Increases Erythrocyte Turnover and Decreases HbA1c - A Population-Based Study” is highly clinically relevant for interpreting blood analyses in individuals at risk of diabetes and reveal important correlations between the HFE hereditary hemochromatosis gene polymorphism, reticulocyte parameters (count and erythrocyte renewal time), and a decrease in HbA1c levels.

It is not clear while these findings appear predictable, what was the specific necessity of confirming them and what novel insights do they provide for scientific understanding and clinical practice. The study's primary objective regarding this specific genetic disorder could be clearer. Please elaborate on the rationale for studying HFE hemochromatosis, and its general epidemiological prevalence.
Beyond HFE, hemochromatosis is caused by mutations in other genes (HJV, TFR2, SLC40A1, CP). Please justify the primary focus on HFE in this study.

Please, clarify data interpretation:in Table X, certain values (e.g., Hb = 14.1 across all genotype columns) appear identical, yet statistical significance is reported (P < 0.001). Could you please clarify this apparent discrepancy?

Additionally, the reported iron level of "14" for all normal genotypes seems uniformly low. Please comment.
The data on smoking status is presented but not accounted for in the analysis. What was the rationale for its inclusion?

Please expand the introduction to include the frequency of the C282Y/C282Y genotype in other populations, if known.
:How was the substantial cohort assembled? Is the C282Y/C282Y test commonly listed in laboratory panels, and how frequently is it ordered in clinical practice?
Methods:
- please justify the chosen methodology for measuring oxidative stress; provide a reference for the method used to determine oxidative stress in urine
- Non-fasting laboratory analyses were used. Could this have introduced bias, particularly for parameters like glucose? Was fasting plasma glucose data unavailable or considered?
- a methodological concern arises regarding sample timing: blood was collected between 2010-2013, while spot urine samples were taken at inclusion. How does the study account for potential temporal variability when comparing these samples?
Results:For the genotypes listed (H63D/NC, H63D/H63D, C282Y/NC, C282Y/H63D, C282Y/C282Y), the text states that iron levels consistently increase. Could you elaborate on the biological or statistical basis for this progression?
The mention of "two Danish prospective studies" lacks context. Please provide their identifiers (e.g., cohort names, registration numbers) and summarize their primary goals and outcomes relevant to this work.
A dedicated "Conclusion" section is absent. What is the primary practical or clinical takeaway from this research?
The Supplementary Materials referenced are currently inaccessible.

Comments for author File: Comments.pdf

Author Response

Reviewer-2

Dear Authors,

Answer: Thank you very much for carefully reading our manuscript and providing valuable feedback. We have answered all your questions, and made changes to the manuscript accordingly.

It is not clearwhile these findings appear predictable, what was the specific necessity of confirming them and what novel insights do they provide for scientific understanding and clinical practice. The study's primary objective regarding this specific genetic disorder could be clearer. Please elaborate on the rationale for studying HFE hemochromatosis, and its general epidemiological prevalence.

Answer1. Thank you for asking this question. In GWAS articles a myriad of significant findings come up, but some of these, particularly the striking finding of decreased erythrocyte counts with C282Y (ref), was not mentioned at all in the main articles. The finding was placed in a supplementary section with several Excel sheets with results from hundreds of genes. Thus, there has been no attention to this finding, which made us curious as to why the count was lower, when in fact, you would think it was higher, because the indices are higher. Combined with the also counter-intuitive finding that HBA1c is decreased in individuals with C282Y despite the fact that they have a higher risk of diabetes, it made us ask the question that maybe this could be explained by lower erythrocyte counts and/or higher reticulocyte counts. No one has asked these questions before. And while our findings for the erythrocyte indices are not novel, we combine these findings with a highly clinically relevant question: why is HbA1c lower, when you would think it was higher in C282Y due to their increased diabetes risk? In addition, we found other relevant articles that support this, such as the findings of oxidative stress and iron overload with a rigid erythrocyte membrane (ref), and the findings of increased phosphatidylserine across all HFE genotypes (ref), which also support the finding of premature aging of the erythrocytes.

We have therefore clarified that in the introduction.

Beyond HFE, hemochromatosis is caused by mutations in other genes (HJV, TFR2, SLC40A1, CP). Please justify the primary focus on HFE in this study.

Answer2: we decided to focus on the HFE C282Y, as more than 80% of patients with genetic hemochromatosis are homozygous for this mutation (Feder 1996). Furthermore, non-HFE hemochromatosis (HJV, TFR2, SLC40A1) are much rarer, and their clinical and laboratory phenotypes are very different from the HFE hemochromatosis (Brissot 2018).

We added the following to the introduction: “Hereditary haemochromatosis (HH) is a genetic iron overload disorder. It is caused by homozygosity for the functional missense mutation C282Y (rs1800562[GàA]) in the HFE gene in more than 80% of hemochromatosis patients in Northern European populations (Feder 1996)”.

We added the following to the discussion: “While there are other non-HFE hemochromatosis mutations, these are much rarer and their clinical and laboratory phenotypes are very different from the HFE hemochromatosis (Brissot 2018) and were therefore not included in this study”.

Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo R Jr, Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel BE, Kronmal GS, Lauer P, Lee VK, Loeb DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morikang E, Prass CE, Quintana L, Starnes SM, Schatzman RC, Brunke KJ, Drayna DT, Risch NJ, Bacon BR, Wolff RK. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet. 1996 Aug;13(4):399-408

Brissot P, Pietrangelo A, Adams PC, de Graaff B, McLaren CE, Loréal O. Haemochromatosis. Nat Rev Dis Primers. 2018 Apr 5;4:18016

Please, clarify data interpretation:in Table X, certain values (e.g., Hb = 14.1 across all genotype columns) appear identical, yet statistical significance is reported (P < 0.001). Could you please clarify this apparent discrepancy?

Answer: Because the N is so large even very small differences on the second or third decimals (not shown as otherwise the tables would become massive) will result in statistical differences.

Additionally, the reported iron level of "14" for all normal genotypes seems uniformly low. Please comment.

We doubled-checked the result for iron and the value is 13.8 for non-carriers (Table 1), 13.9 in CGPS (Suppl table 2), and 13.1 in GESUS (Suppl table 3).

The data on smoking status is presented but not accounted for in the analysis. What was the rationale for its inclusion?

Answer: Reviewer-1 also made a similar comment. We have now presented a supplementary table with results from a sensitivity analysis including confounders that are not part of the pathway from genotype->iron->biomarker. In a stepwise regression, the model includes age, sex, genotype, cohort, blood donor status, BMI, alcohol consumption, smoking, physical activity, and infection. Results were similar with or without these covariates in the model. We have also added a paragraph to the statistical section explaining this. And we have commented on this in the results section “Sensitivity analyses”. Lastly, we have included information on alcohol, physical activity, and infection in table 1 and in the supplementary tables of the cohorts.

Please expand the introduction to include the frequency of the C282Y/C282Y genotype in other populations, if known.
- We have added the following to the introduction: “The frequency of the A-allele ranges from 5-10% in Northern European populations (ref), and the prevalence of homozygosity for C282Y (C282Y/C282Y) ranges from 0.30-0.60% in similar populations (ref).”
- We have added the following the results: “The frequency was 5.9% for the A-allele (rs1800562) for C282Y and 13.1% for the G-allele (rs1799945) for H63D (Supplementary Table 1), and the prevalence of C282Y/C282Y (N=399) was 0.32% for both cohorts combined (Table 1).”
- We have added the following to the discussion:” The prevalence of C282Y/C282Y was 0.32% which was similar to another Danish study (0.36%), slightly higher than in Sweden (0.23%), and slightly lower than other populations of Northern European descent, such as in USA and Canada (0.44%), UK (0.50%), and Australia (0.60%).”
- Here is a list of the frequencies with references which are also reported in the discussion:

Answer4: Below, we have provided frequencies for C282Y/C282Y homozygosity in other white populations of Northern European descent, where the mutation is most prevalent.

How was the substantial cohort assembled? Is the C282Y/C282Y test commonly listed in laboratory panels, and how frequently is it ordered in clinical practice?
- The two cohorts were assembled separately in the Copenhagen greater area, and in a suburb 70 km south of Copenhagen. Participants were invited based on their civil registration number and living in these areas at the time of recruitment. Thus, the individuals were participants in population-based studies not patients admitted for iron overload. We have inserted this information in the Methods.
- The C282Y and H63D mutations are part of the investigation panels for iron overload in Denmark; in clinical practice, the genotype is requested if individuals have a transferrin saturation consistently above 45%. We do not know how frequently it is ordered. In our study, all participants were genotyped regardless of transferrin saturation. We added the following to the discussion: “The unique aspect of the included cohorts is that all participants were genotyped for C282Y and H63D and had transferrin saturation measured, as opposed to the routine clinical practice which would be to genotype only those individuals with transferrin saturation above 45% (ref)”

Answer

Methods: please justify the chosen methodology for measuring oxidative stress; provide a reference for the method used to determine oxidative stress in urine
- Answer 6. We have now provided a reference for the method. Furthermore, we have added a justification for the choice of method with a reference to the European Standards Committee on Urinary (DNA) Lesion Analysis: “The choice of LC-MS/MS was based on high specificity for these analytes and speed of analysis (refs)”
- Answer 6. We inserted the following sentence as a limitation in the discussion: “We did not measure fasting glucose. However, since the estimated average glucose (EAG) over the erythrocyte lifespan is always based on the HbA1c which is always measured non-fasting, we believe that the measured non-fasting venous plasma-glucose was more comparable to the EAG, than if we had measured fasting glucose.”
- Answer: Urine and blood samples were collected on the same day and time. The measurements of the urine samples were a couple of years later than collection as the samples were biobanked before measurement. Since the LC-MS/MS method is quite costly and to avoid batch effects, we measured the samples within a short period of time. We have now clarified the sentence: “In GESUS, spot urine samples were collected at baseline on the same day as the blood collection and biobanked for 1-3 years at -80^oC before measurement”

Non-fasting laboratory analyses were used. Could this have introduced bias, particularly for parameters like glucose? Was fasting plasma glucose data unavailable or considered?
a methodological concern arises regarding sample timing: blood was collected between 2010-2013, while spot urine samples were taken at inclusion. How does the study account for potential temporal variability when comparing these samples?

Results: For the genotypes listed (H63D/NC, H63D/H63D, C282Y/NC, C282Y/H63D, C282Y/C282Y), the text states that iron levels consistently increase. Could you elaborate on the biological or statistical basis for this progression?

Answer 7. We inserted the following in the methods section about the order of the genotypes: “It is well established that C282Y/C282Y disrupts the HFE protein and this is called HFE hemochromatosis (or Type1A) when iron overload and symptoms arise (Kowdley 2019). H63D is a modifier to the heterozygous C282Y (compound heterozygosity) and this genotype is also associated with iron overload and hemochromatosis although the penetrance is lower than for C282Y/C282Y(Kowdley 2019). There is more debate about the penetrance of hemochromatosis with C282Y/NC and H63D/H63D, but these variants confer mild iron overload, and the H63D/NC is generally considered a low penetrance variant(Kowdley 2019). ”
We changed the statement in Results so it now reads: “Individuals with C282Y/C282Y had elevated plasma iron concentration, transferrin saturation, ferritin concentration hematocrit, hemoglobin, MCV, and RDW, and there was an increasing trend across the HFE genotypes (H63D/NC, C282Y/NC, H63D/H63D, C282Y/H63D, C282Y/C282Y). Individuals with C282Y/C282Y and C282Y/H63D had increased 8-oxoGuo; C282Y/C282Y had increased hsCRP, and all genotypes had increased ALAT compared to non-carriers (Table 1).”

The mention of "two Danish prospective studies" lacks context. Please provide their identifiers (e.g., cohort names, registration numbers) and summarize their primary goals and outcomes relevant to this work.

Answer 8. We mention the cohort names of the studies in the following sentences. Neither study has a registration number, as neither were trials. We have now provided the local IRB approval numbers instead.
The goals for the GESUS study has previously been described in PMID:24001461 and the goal of the CGPS were similar: “The aim of GESUS is to facilitate epidemiologic and genetic research by using information from questionnaires, health examinations, biochemical measurements, genetic variants and public registers to analyze the occurrence of co-morbidities (e.g. diabetes, cardiovascular disease, pulmonary disease and cancer) and mortality.” (this study is also referenced in the article).

A dedicated "Conclusion" section is absent. What is the primary practical or clinical takeaway from this research?

Answer 9. There was not a dedicated section by the strict IJMS template. The conclusion is mentioned in a separate paragraph at the end of the Discussion. The takeaway is mentioned in the very last sentence of the manuscript which is the last sentence of the conclusion: “Thus, HbA1c may not be a useful biomarker for diabetes diagnosis in individuals with C282Y/C282Y.”

The Supplementary Materials referenced are currently inaccessible.

Answer 10. The supplementary material was submitted. We are sorry you missed it.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank the authors for making the suggested modifications. I consider the article accepted.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors corrected inaccuracies in the tables, clarified the research objective, and improved the overall clarity. Following these revisions, the article became more informative, though it remains quite lengthy

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C282Y Homozygosity Increases Erythrocyte Turnover and Decreases HbA1c—A Population-Based Study

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